Monitoring protein-protein interactions between the mammalian integral membrane transporters and PDZ-interacting partners using a modified split-ubiquitin membrane yeast two-hybrid system.
PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available ... to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this "MYTH 2.0" system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes.
Mesh Terms:
Animals, Cells, Cultured, Cloning, Molecular, Female, Humans, Mammals, Membrane Proteins, Membrane Transport Proteins, Mice, Models, Biological, Oocytes, PDZ Domains, Plasmids, Protein Binding, Rats, Saccharomyces cerevisiae, Two-Hybrid System Techniques, Ubiquitin, Xenopus
Animals, Cells, Cultured, Cloning, Molecular, Female, Humans, Mammals, Membrane Proteins, Membrane Transport Proteins, Mice, Models, Biological, Oocytes, PDZ Domains, Plasmids, Protein Binding, Rats, Saccharomyces cerevisiae, Two-Hybrid System Techniques, Ubiquitin, Xenopus
Mol. Cell Proteomics
Date: Jul. 01, 2008
PubMed ID: 18407958
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